The invention relates to caliciviruses, which are newly recognized as potentially important human pathogens. In a population of adult blood donors from Oregon, 18% were found to exhibit caliciviruses-specific antibodies.
Viruses of the family Caliciviridae are non-enveloped single stranded positive sense RNA viruses having a genome of about 8 Kbs in size. The capsid protein is a single molecular species of about 60 KDa and forms 22 cups or calyxs on the viral surface thereby providing the morphologic basis for the name Caliciviridae. Viruses of the calicivirus family have been known to be pathogenic in mammals since at least 1932 when they were shown to be the causative agent of vesicular exanthema of swine (VES). Subsequently, caliciviruses have been shown to be pathogens for cats and marine mammals, such as sea lions.
The family of Caliciviridae is divided into four taxonomic genera: Norwalk viruses and Sapporo viruses (both of which are gastrointestinal pathogens in humans), rabbit hemorrhagic disease viruses and vesiviruses. The first three of these genera all have proven refractory to in vitro cell culture replication. The fourth genus, the vesiviruses, may be replicated in cell culture, for instance using Vero cells. Despite the fact that caliciviruses have been known for many years as pathogens of mammals, cultivatable caliciviruses have not been found to be of clinical significance in the human population. In particular, vesiviruses have not shown to be significant infectious agents for humans.
Serologic evidence can suggest that animal viruses are also human pathogens and occasionally this is confirmed by a single case as was reported for Cache Valley virus [3]. Other times, with no prior warning, a single case signals the arrival of a new zoonotic disease as occurred with the death of a horse trainer in Australia infected with a previously unknown morbillivirus [4]. Such events can occur when shifts in ecologic relationships cause intermixing of species and result in increased xe2x80x9cviral trafficxe2x80x9d [5]. In turn, xe2x80x9cviral trafficxe2x80x9d encourages adaptive shifts in viral genomes and host-parasite relationships which can result in emerging new diseases, especially zoonoses [5]. An increase in viral traffic for one calicivirus has been deliberate in Australia, where hemorrhagic virus of rabbits [6, 7, 8] has been spread as a biologic control agent, resulting in a continent-wide calicivirus challenge of naive and diverse populations.
The early detection of, and appropriate responses to, emerging viral diseases that may result from events that heighten viral traffic require preventive programs involving research, early diagnosis, public health awareness and reporting.
The invention concerns reagents and methods of detecting calicivirus in humans. The invention also provides calicivirus nucleic acid sequences and amino acid sequences that are useful for developing immunostimulatory polypeptides. As will be made clear by the specification, calicivirus infection is linked to human diseases, reproductive dysfunctions, and moreover calicivirus in humans may be transmitted via blood.
It has been discovered that caliciviruses are common infective agents in humans and can cause a serious vesicular disease characterized by fever, generalized myalgia and painful blistering of the hands and feet and a new strain of calicivirus (belonging to the vesivirus genus), that is called xe2x80x9cSan Miguel sea lion virus 5 serotype Homosapien-1xe2x80x9d (SMSV-5 Hom-1) has been discovered and isolated. This disclosure is directed towards methods of detecting an infective agent in a sample from a human comprising detecting calicivirus antigens and/or anti-calicivirus antibodies; to methods of stimulating immune responses in human subjects against caliciviruses; and to SMSV-5 Hom-1, its polynucleotides and polypeptides.
One aspect of the present invention is a method of detecting a past (or present) infection of a human by an infectious viral agent comprising detecting anti-calicivirus antibodies in a sample from the putatively infected human subject. Another aspect of the present invention is a method of detecting a present viral infection of a human comprising detecting one or more calicivirus antigens and/or calicivirus genomic segments in a sample taken from the putatively infected human subject. A further aspect of the present invention is the provision of a protective vaccine against caliciviruses comprising inoculating a human subject with a vaccine comprising at least one calicivirus antigen or its cDNA sequence with a vector comprising a recombinant caliciviral polynucleotide wherein the viral vector expresses at least one calicivirus antigen.
Another aspect relates to nucleotides of SMSV-5 Hom-1, some of which may be used as probes or primers, for instance for use in detecting or amplifying viral polynucleotides. Yet another aspect relates to proteins of SMSV-5 Hom-1, which may be antigenic, some of which may be used in diagnostic and immune stimulatory applications such as the creation of vaccines. The invention also relates to antibodies specific for SMSV-5 Hom-1, which in certain embodiments may be monoclonal antibodies. Also included are assays to detect antigens of SMSV-5 Hom-1. Additionally, the invention includes assays to detect antibodies specific for SMSV-5 Hom-1. A further aspect of the invention relates to vaccines against SMSV-5 Hom-1 infection. All these different embodiments are discussed in detail below.
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
SEQ ID NO: 1 shows the nucleic acid sequence of the SMSV-5 Hom-1 RNA polymerase region.
SEQ ID NO: 2 shows the amino acid sequence of the SMSV-5 Hom-1 RNA polymerase region.
SEQ ID NO: 3 shows the nucleic acid sequence (1671 bases) of the p5RT73 genomic segment of SMSV-5.
SEQ ID NO: 4 shows a cDNA that encodes an amino acid sequence that is useful for detecting caliciviral specific binding agents.
SEQ ID NO: 5 shows the amino acid sequence encoded by the cDNA of SEQ ID NO: 4.
SEQ ID NOS: 6-10 show PCR primers.
SEQ ID NOS: 11-13 show the three possible ORFs of the nucleic acid sequence shown in SEQ ID NO: 3.
SEQ ID NO: 14 shows the nucleic acid sequence of a 300 bp probe that can be used to detect a broad range of caliciviruses.